1. Field of the Invention
The present invention relates to an electron microscope which can irradiate an electron beam onto a specimen and detects an electron transmitting the specimen or a secondary electron and a reflected electron emitted from the surface of the specimen to obtain a magnified image. More specifically, the present invention relates to a bio electron microscope.
2. Description of the Related Art
To observe, in particular, a bio specimen using an electron microscope, there are fine structure analysis of tissues, structure analysis of protein, and examination of virus. In recent years, various diseases including bovine spongiform encephalopathy (BSE), communicable disease such as HIV and HIC, food poisoning due to O157, and carbon fungus terrorism have been increased. Needs for high-reliability analysis and examination by a highly-magnified image observation using an electron microscope are on the increase. When noted virus and protein are fine to require high resolution and it is desired to observe their inner structures, a transmission electron microscope (TEM) performing imaging by an electron transmitting a specimen is used. A bio TEM is described in the H-7600 transmission electron microscope catalog No. HTD-040 (issued in November 2001) of Hitachi High-Technologies Corporation. It has an electron gun, an illumination system electron lens, a specimen holder and stage, an imaging system electron lens, a camera, an evacuation system, and a control system. Electron accelerating voltages for a bio TEM are mainly 100 kV and 120 kV, and may be used by lowering it to about 50 kV. A bio specimen observation procedure by the transmission electron microscope is described in “Electron Microscope”, Vol. 37, No. 2, p. 81-84 (2002). In the examination of virus, a small round structured virus having a diameter of several tens of nm causing food poisoning is purified and concentrated from a fecal matter to observe an image magnified tens of thousands of times to hundreds of thousands of times, and then, the presence or absence of virus and the species of the virus are decided from its shape and the characteristic of its inner structure. In addition, in the structure analysis of protein, several magnified images of a protein specimen extracted and purified from a tissue are observed by tilting a specimen stage of the electron microscope, and then, the images are subjected to CT (computed tomography) processing to obtain a fine three-dimensional structure in the size of several tens of nm.
Bio specimens used for the transmission electron microscope are broadly divided into three: {circle around (1)} a stained section specimen, {circle around (2)} a negative stained specimen, and {circle around (3)} a frozen section specimen. In the stained section specimen, the tissue of an animal or plant is cut into a section having a thickness of several tens of nm using a microtome equipped with a blade of diamond or glass, which is then placed on a meshed thin plate for electron microscope. To make the section, there are processes such as fixation, anhydration, embedding and cutting of the tissue. To obtain contrast corresponding to the tissue structure in an electron microscope image, staining the specimen is generally needed. As a stain, a reagent including heavy metal such as uranium acetate, lead citrate, lead hydroxide, or lead acetate is used. In general, the specimen is double-stained by uranium and lead. The reason why staining is needed is that since a main element constituting a living body are light elements such as hydrogen, oxygen, carbon and nitrogen, those scattering factors to an electron beam and its difference between the elements are small so that image contrast is very hard to provide. Stained is protein in the tissue. The higher the concentration of protein, the stronger protein is stained. As a result, contrast dependent on the concentration of protein is obtained in the electron microscope image.
In the {circle around (2)} (negative stained specimen, a tissue or fecal matter purified and concentrated using a reagent and a centrifugal machine is placed on a meshed thin plate for electron microscope. A particulate specimen such as virus is a representative example. As a stain, a phosphorate tungsten acid (PTA) liquid is generally used. In this method, in the case of virus, a bank of the stain is formed around the virus to form contrast between the bank and the virus. In the {circle around (3)} frozen section specimen, a tissue is brought into contact with a copper block cooled by liquid helium or liquid nitrogen to be instantaneously frozen, and is then cut in a frozen state using a microtome equipped with a cooling stage. Observation of a tissue structure in an active state is aimed. Fixation and staining are not conducted, and a cryo-electron microscope equipped with a cooling specimen stage is used for observation. Since the specimen is not stained, the image contrast is very low.
In the recent transmission electron microscopes, a lens current, a shutter, and a specimen stage are controlled by a PC (personal computer) to facilitate operation such as focusing, photographing, and specimen slight movement. They have functions of automatic focus, automatic photographing, and automatic montage (obtaining a wide area image joined by automatically repeating specimen slight movement and photographing). The electron microscope is not equipped with functions of specimen preparation such as extraction, purification, concentration and staining to observe a specimen by the electron microscope, and of analyzing the species of virus and a protein three-dimensional structure from a photographed image.
The bio specimen observed by the transmission electron microscope is susceptible to damage by electron irradiation. A constituent constituting the tissue contains about 85% water. It also contains 10% protein, 2% adipo, 1.5% mineral, and 1.1% nucleic acid which are weak to electron irradiation. When an electron beam having an accelerating voltage of 100-120 keV is irradiated onto a specimen of a tissue of such structure, damage such as deformation, decomposition and destruction of the fine structure occurs. The stained section specimen and the negative stained specimen stained by heavy metal are rather stronger to electron irradiation than the frozen section specimen. The damage cannot be avoided as an essential problem. With the damage, the bio fine structure cannot be correctly analyzed and the accuracy of the examination is lowered. In particular, when a three-dimensional structure of a cell tissue is observed by TEM, the same area of the specimen is irradiated several tens of times while tilting the specimen. The electron dose is very large to make the damage significant. In this case, since the structure is gradually changed during photographing, the images at the respective tilt angles do not reflect the same structure. When image processing of the three-dimensional reconstruction is conducted using the images, artifact occurs without obtaining any correct three-dimensional structures.
In addition, unless the bio specimen is stained as described above, high contrast cannot be obtained. The fine structure may be changed by staining. It may not be understood that how the structure observed using the stained specimen corresponds to the original structure. These cause erroneous analysis and lowered examination accuracy.
The prior art electron microscope basically observes a magnified image of fine structure. Understanding the image, that is, extracting necessary information must be conducted by a user. A typical pathologic diagnosis by the electron microscope includes specification of viral disease. The species of virus is specified from the structure characteristic of a viral image observed by the electron microscope. For that, high knowledge and experience are needed. Only a limited number of scientists can conduct this.
The recent electron microscopes are equipped with image processing software. It can subject fast Fourier transformation to an image photographed by a CCD camera for frequency component analysis and conduct grain size analysis. It, however, is only placed supportably in order that the user can extract necessary information.
Further, in the preparation of the bio specimen, as described above, many processes such as fixation, anhydration, embedding, cutting and staining of the tissue are manually conducted for a long time. The respective processes must be optimum for each desired tissue structure for each specimen, requiring high technology, know-how and experience. Depending on variation in the preparation condition, quite different electron microscope images of the same tissue may also be obtained.